The binding protein concentration should be at least 10 times that required for routine assays to ensure PO > 0.9. The range of ligand concentrations chosen would also need to be increased so that a complete inhibition curve is traced. This may require a partial purification and concentration of the inhibitor but does not require its isolation. The label concentration is kept low to avoid selfcompetition when the curve of an unknown substance is being followed. As the binding site concentration is high, this would not mean a decrease for most assays in the number of counts available. Heterogeneity could also interfere with the sharpening of the curves that is the basis of discrimination in the homogeneous case. Unless heterogeneity had its chance discriminatory effect, this could make the proof of identity more uncertain. It is recommended that the inhibition curves should be examined a t several different ro values to ascertain whether the curves obtained are typical of homogeneous or heterogeneous binding.
tem, different ligands may give c values differing by a factor of two (7).So relatively large differences in slope might be expected even in the region of small ro where the homogeneous system would have exhibited relatively small differences. Given an extreme heterogeneity of the binding affinities, a n incomplete inhibition could result. This is equivalent to a resolution of binding into discrete classes of association constant. Incomplete inhibition may give a good discrimination if the label and competitor differ in this respect, as the upper bound is a well-defined property of the inhibition curve.
CONCLUSIONS Lack of identity between inhibitors can be verified by differences of shape of the inhibition curves when the system is homogeneous with respect to affinity constant. The interpretation of shape in a log concentration scale does not require prior knowledge of absolute concentration of the inhibitors. The method is therefore suitable for examination of unknown inhibitors present in biological fluids and in vitro systems. Dilutions should be done as far as possible in a medium similar to the test medium but lacking the inhibitory substance (e.g., control culture medium or the plasma of normal healthy individuals).
Received for review September 5, 1973. Accepted January 11, 1974. The investigation was supported by the Medical Research Council (Grants 970/656/B and 971/817/B).
Support-Bonded Phases for Gas Chromatography Derived from Alkyl and Phenyl Chlorosilanes A.
H. AI-Taiar, J. R. Lindsay Smith, and D. J. Waddington
Department of Chemistry, University of York, Heslington, York YO7 5DD, England
In a previous paper ( I ) , we described the preparation of some thermally stable gas-chromatographic packing materials which were prepared by the hydrolytic polymerization of trichlorosilanes with or without added silicon tetrachloride. The materials which contained a considerable excess of the chlorosilane (98%) allowed the elution of a wide range of nonpolar and polar compounds and were thermally stable up to a t least 350 “C; however, their efficiencies were less than those obtained for many conventional columns. In another approach to making useful stable chromatographic phases, reported first by Abel, Pollard, Uden, and Nickless (2) and investigated in some detail by Aue and Hastings (3-8) and others (9-I2),chlorosilanes are poly(1) A. H. AI-Taiar, J . R. Lindsay Smith, and D. J. Waddington, Anal. Chem., 42,935 (1970). (2) E. W . Abel, F. H . Pollard, P. C. Uden, and G.Nickless, J. Chromatogr., 22,23 (1966). (3) W . A . A u e and C. R. Hastings, J. Chromatogr., 42,319 (1969). (4) C. R. Hastings, W. A. Aue, and J. M. Augi, J. Chrornatogr.. 53,487 (1970). (5) W. A . Aue, C. R. Hastings, J . M . Augl, M . K . Norr, and J . V. Larsen, J. Chromatogr., 56,295 (1971). (6) C. R. Hastings, W. A. Aue, and F. N . Larsen, J. Chromatogr., 60, 329 (1971). (7) W . A . Aue and P. M. Teii, J. Chromatogr., 62,15 (1971). (8) W. A . Aue, S. Kapila, and C. R. Hastings, J. Chromatogr., 73, 99 (1972). (9) H. N . M. Stewart and S. G . Perry, J . Chrornatogr., 37,97 (1968). (10) J. B. Sorrel1 and R. Rowan, Jr.,Anal. Chern., 42,1712 (1970). (11) R. Rowan, J r . , and J. B. Sorrel1,Anal.Chem., 42, 1716 (1970). (12) M. Novotny, S. L. Bektesh, K . B. Denson, K . Grohmann. and W. Parr, Anal. Chem., 45,971 (1973).
merized on the surface of typical chromatographic supports. The materials obtained from this procedure are potentially useful in both gas and liquid chromatography, the organic phase being chemically or strongly physically bonded to the inorganic support. Apart from the obvious advantage of thermostability that these bonded phases have over conventional chromatographic materials, it should be possible to produce a wide range of closely related materials to study solute-solvent interactions in chromatography. In this paper, we describe the preparation of a number of chromatographic materials prepared from phenyl- and substituted-phenylchlorosilanes (and, for comparison, octadecyltrichlorosilane) on Gas Chrom Q as the inert support.
EXPERIMENTAL Operating Conditions of the Gas Chromatograph. All results were obtained using glass columns (1.5-m x 4-mm i.d.) in a Pye 104 gas chromatograph equipped with a flame ionization detector coupled to an RE 511 Goertz Servoscribe recorder. The carrier gas, nitrogen (BOC, “oxygen free”), was deoxygenated and dried by passing it through successively a solution of chromium(I1) chloride in hydrochloric acid over zinc amalgam, concentrated sulfuric acid, potassium hydroxide pellets, silica gel, and molecular sieve 5A. Measurement of Thermal Stability of Packing Materials. The detector ionization current was measured for columns packed with the new materials and with Celite (AW)-silicone elastomer E301 over a wide range of temperature, and compared with that of a n empty column under identfcal conditions. A current of A gave a full scale deflection when the recorder was set at 10 mV with the amplifier attenuation a t 1 x 103. ANALYTICAL CHEMISTRY, VOL. 46, NO. 8, JULY 1974
1135
a
(a)
I
05
I
2
6
-U. cm.4sec:'
Figure 3. Relation of HETP and linear velocity of the carrier gas, nitrogen, for dodecane on column O D (prepared from octaddcyltrichlorosilane and Gas Chrom Q) at 200 OC
(b)
150
100
200
226
me
Table I. A m o u n t of Organic Compound Present on the Column Packing Materials Prepared from Trichlorosilanes and Gas Chrom Q
Figure 1. Mass spectra of hexadecane ( a ) By direct insertion. ( b ) 0.08 pl hexadecane obtained after passage through the column Ph (prepared from phenyltrichlorosilane and Gas Chrorn Q ) at 325 "C
Abbreviation of column material used subse- % ' (w/w)organic quently material
Trichlorosilane
Phenyl
Ph
p-Tolyl p-Methoxyphenyl p-Bromophenyl p-Trichloromethylphenyl Octadecyl
To1
17.7 18.1 13.9 22.4 20.3 18.2
MP BP TCT OD
Table 11. Flame Ionization Detector Currents Obtained for the Column Packing Materialsa 150
100
200
226
mie
Figure 2. Mass spectrum of hexadecane
Columna: CelitePh To1 MP BP silicone elastomer E301 Tempera(2O%w/w) 10'0 X Flame ionization detector current, A ture, OC
0.08 pI hexadecane obtained after passage through column BP (prepared from p-bromophenyltrichlorosiiane and Gas Chrom 0)at 325 "C. The mass spectrum of hexadecane obtained by direct insertion is given in Figure 1 (a)
The mass spectra of materials following gas chromatographic elution between 300-350 "C were compared with the spectra obtained from the same materials by direct insertiop. For these experiments an A.E.I. MS 12 mass spectrometer was used with a 70-eV electron energy, 100-pA trap current, 8-kV accelerating potential, and a sourcetemperature of 165 ' C . For one column packing, namely that derived from p-bromophenyltrichlorosilane, the change in carbon content was measured after the material had been heated at 350 "C for nine days in a stream of oxygen-free nitrogen. Measurement of Retention D a t a a n d Column Characteristics. All the retention data recorded were obtained using a carrier gas flow rate of 20 cm3 min-l, and retention times ( t ' d are adjusted to allow for the gas hold-up time. The efficiency of a column as represented by an HETP-carrier gas linear velocity curve was measured using dodecane as the standard solute. A selection of aliphatic and aromatic compounds with different electronic and structural characteristics was chosen as solutes to examine the influence of structural changes in the organic moiety of the stationary phases on their chromatographic properties. Materials. Trichlorosilanes Used t o Prepare Packing Materials. p-Bromophenyl-, p-methoxyphenyl-, and p-tolyltrichlorosilanes were prepared from magnesium, silicon tetrachloride, and p-dibromobenzene, p-bromoanisole, and p-bromotoluene, respectively, by methods due to Petrov et a2. (13, 14). p-Trichloromethylphen-. yltrichlorosilane was prepared by a method adapted from that of Frost ( 1 5 ) , chlorine being passed through p-tolyltrichlorosilane under the influence of UV light a t 95-100" for 54 hours. The tri'
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ANALYTICAL CHEMISTRY, VOL. 46, NO. 8 , JULY 1974
100 150 200 224 250 275 300 320 340 360 a
0.2 0.5 1.4 3 .I 9 .o 23.8
0.0 0.0 0.0
...
...
... ...
0.0
0.0 0.0
0.0 0.0
0.0
0.1
0.3
0.2
...
...
0.0
0.0 0.3 0.9 2.2
1.0
0.0
0.0 0.0
0.0 0.0 0.1
...
...
0.0
0.0
0.0 0.1 0.4 1.0
. . .b
...
OD
...
. . I
1.8
0.2
3.7 9.4
0.4 1.2
...
... 1.0 1.5 3.4 8.3
Linear Velocity of the carrier gas, 4.0 cm sec-1. *Results not obtained.
chlorosilanes were purified by fractional distillation, Phenyl- and octadecyl-trichlorosilanes were obtained commercially (Hopkin and Williams Ltd.). Preparation of Packing Materials. A series of gas-chromatographic packing materials was prepared from the trichlorosilanes described above in the presence of an inert solid, Gas Chrom Q (obtained from Applied Science Laboratories). A trichlorosilane was dissolved in an inert solvent, sodium dried light petroleum ether (bp SO-SO"), and added to Gas Chrom Q.After the mixture had been shaken for 5 hr in a closed vessel, the solvent was removed under reduced pressure. A stream of warm air saturated with water vapor was then passed over the powder until no more (13) A. D. Petrov, V. A. Ponomarenko, 6. A. Sokolov. and V . U. Roshal, J. Gen. Chem. USSR, 26, 1391 (1956). (14) A. D. Petrov, M . I . Batuev, V . A. Ponomarenko, A. D. Snegova, A . D. Matveeva, and B. A. Sokolov, J. Gen. Chem. USSR, 2 7 , 2112 (1957). (15) L. W . Frost, J . Amer. Chem. SOC., 7 8 , 3855 (1956).
Table 111. Retention Times (adjusted, tr', (min), and relative to that obtained for Pentane, Rt,') for Some Alkanes and Alkenes on Columns Ph and TCT (Prepared from Phenyl- and p-Trichloromethylphenyl-trichlorosilanes and Gas Chrom Q) a Ph column at SOo Solute
Propane Butane 2-Methylpropane Pentane 2-Methylbutane 2,2-Dimethylpropane Hexane 2,2-Dimethylbutane Propene Butene-1 cis-Butene-2 2-Methylpropene Pentene-1 Pentene-2 2-Methylbutene-2 Hexene-1 3,3-Dimethylbutene-1 2,3-Dimethylbutene-2 Cyclopentane Cyclohexane Cyclopentene Cyclohexene a
t,' (min)
0.9 3.3 2.1 11.5 9 .o 2.2 40.3 11.5 0.9 3.4 4.5 3.3 11.5 13.7 14.8 37.6 11.8
54.1 17.2 52.2 16.7 69.8
Rtr'
78 287 183 1000
783 191 3504 1000
78 296 391 287 1000 1191 1287 3270 1026 4704 1496 4539 1452 6070
TCT column at 150' Rt,' on Ph t,'(min)
Rtr' Rtr' onTCT
1.3 76 4 . 8 282 3 . 7 218 17.0 1000 15.2 894 9 . 7 571 57.7 3394 37.1 2182 1.2 71 4 . 3 253 5 . 0 294 4 . 3 253 15.5 912 1 7 . 1 1006 17.4 1023 52.7 3100 31.2 1835 56.3 3312 16.9 994 56.4 3318 1 5 . 3 900 62.3 3665
1.03 1.02 0.84 1.00
0.88 0.33 1.03 0.46 1.10 1.17 1.33 1.13 1.10
1.18 1.26 1.05 0.56 1.42 1.51 1.37 1.61 1.66
Table IV. Adjusted Retention Times, t,' (min),for Some Aromatic Compounds on Stationary Phases Prepared from Trichlorosilanes and Gas Chrom Qa Solute
o-Xylene m-Xvlene p-xylene Aniline o-Chloroaniline m-Chloroaniline p-Chloroaniline o-Cresol m-Cresol p-Cresol o-Nitrotoluene m-Nitrotoluene p-Nitrotoluene Chlorobenzene Bromobenzene Iodobenzene
(16) D M Ottenstein, J Gas Chrornatogr, 6 129 ( 1 9 6 8 )
1. I 1.9 3.8 3.2 3.1 7.4 15.6 23.8 23.9 8.2 8.8 8.8 18.0 20 . O 21.2 3 .O 5.0 9.7
To1
MP
BP
2.0 3.7 7.7 6.6 6.5 13.1 31.3 51.6 49.7 15.9 17.1 17.2 40.5 45.5 46.7 6.0 10.1 19.7
2.5 4.8 10.6 8.8 8.5 24.1 51.1 85.2 93.3 34.0 40.1 38.8 66.2 75.4 83.3 7.6 13.3 27.0
1.3 2.6 5.9 4.8 4.7 8.9 20.4 29.6 30.3 10.7 11.9
TCT
4.5 10.9 28.3 23.9 24.0 26.4 ... ... ...
...
...
12.1
...
29.4 39.9 47.4 4.1 7.2 15.1
183.1 254.4 308.2 17.2 30.5 68.5
a Carrier gas, nitrogen, 20 om3 min-1; glass column, 1.5-m X 4-mm id 200oc.
Table V. Adjusted Retention Times Relative to Benzene, Rt,', of Some Organic Compounds on Columns Prepared from Trichlorosilanes and Gas Chrom Q at 325°C" Solute
Carrier gas, nitrogen, 20 cm3 min -1; glasa column, 1.5-m X 4-mm id.
hydrogen chloride was detectable in the effluent. The solid was initially dried a t 150" for 12 hr under deoxygenated nitrogen and then at 370" for 60 hr in a stream of the nitrogen. The solid was finally treated with dimethyldichlorosilane (Hopkin and Williams Ltd.), using the method due to Ottenstein (16). The organic compositions of the packing materials are given in Table I. RESULTS AND DISCUSSION The materials obtained by the hydrolysis of a trichlorosilane in the presence of the inert support Gas Chrom Q show a high thermal stability. For example, the loss of. organic material from the material prepared from the p bromophenyltrichlorosilane, heated in a stream of nitrogen at 370" for nine days, was very small with a loss of 0.14% in carbon. The thermal stability of the packing materials was examined in more detail by two methods. First, the flame ionization detector current was recorded by comparison us an empty column over a wide range of temperatures and this was assumed to be a measure of the column bleed (Table II). A comparison of these results with those from the silicone elastomer column and the column prepared from the cohydrolysis of silicon tetrachloride and octadecyltrichlorosilane (column B4(e)) ( I ) shows the superior thermal stability of materials in the present study. The standard silicone elastomer (20% w/w E301) column was over three years old and before comparative studies were made it was heated a t 100 "C for 72 hr, and 200 "C for 48 hr, and finally a t 240 "Cfor 2 hr. Second, using hexadecane as the standard solute, mass spectra obtained by direct insertion were compared with those obtained following chromatographic elution at temperatures between 300 and 350 "C. Figures 1 and 2 are examples of these spectra and clearlyshow the low background from column bleed confirming the thermal stabilit y of these materials.
Column: Ph
Benzene
Column: Ph
Dodecane Naphthalene Biphenyl Anthracene Dibenzyl Diphenylmethane 1,8-Diamino-octane N,N-Dimethyltryptamine Dimethylphthalate Benzyl benzoate Anthraquinone Benzophenone n-Dodecanol Fluorenone o-Nitroaniline m-Nitroaniline p-Nitroaniline p-Nitrobenzaldehyde a
1.3 5.7 8.4 31.6 10.5 9.2 4.2 28.5 11.3 15.3 54.9 17.9 3.7 30.0 12.2 16.4 24.9 9.6
To1
BP
OD
2.0 6.6 9.1 38.3 11.6 10.4 4.4 31.5
3.5 8 .o 10.9 46.9 17.2 13.6
2.9 5.2 8.4 30.1 11.o 8.8 4.2 1.9 6.4
12.8
26.1 66.4 20.4 4.5 35.8 18.5 24.6 37.2 14.0
11.2
40.9 14.3 36 . O 103.0 29.4 10.9 49.8 21.8 27.7 41.8 32.5
1.8
43.4 14.5 5.8 23.4 7.6 8.8 11.8 10.7
Carrier gas, nitrogen, 20 cm3 min-1; glass column, 1.6-m X 4-mm id.
The HETP-linear velocity plots from the columns studied were similar. In order to obtain as direct a comparison as possible with the columns prepared earlier, Figure 3 illustrates, the plot for the column prepared from octadecyltrichlorosilane. The minimum is similar to that obtained by Aue and Hastings (3, 4 ) and, indeed, is in the same range as that found for many conventional GLC columns. By using Gas Chrom Q, a solid support that is largely inert (the surface is deactivated by silanization), it is unlikely that the thermal stability of the packing materials arises from chemical bonding of the siloxane to the support. It would appear that the polysiloxanes are intermeshed with the support and, unlike a conventional liquid phase, the organic material cannot be removed effectively by heat. That most of the polysiloxane is on the surface of the support material seems probable from scanning electron microscopy studies ( 5 ) . The materials prepared in this study allow the elution and separation of a wide range of compounds over a large ANALYTICAL CHEMISTRY, VOL. 46, NO. 8, J U L Y 1974
1137
ouste Gulbenkian Foundation for a research scholarship and to the Universities of Baghdad and Basrah for leave of absence.
Supplementary Information for “Reagent Chemicals,” Fourth Edition 0
3
0
3
I 0
TIME irninl
Figure 4. Separation of mixtures of compounds 1, Chlorobenzene; 2, Bromobenzene; 3, lodobenzene; 4, Diphenylmethane on MP at 325 “C. 5, Pentane: 6, Hexane; 7, Heptane; 8. Octane; 9, Nonane; 10, Decane on MP at 200 “C. 11, Diethyl ether; 12, Phenol; 13, Aniline on BP at 200 “C. 14, Hexene-1; 15, Heptene-1; 16, Octene-1 onTol at 180°C Flow rate for all experiments, 20 cm3 cm-
’,
temperature range. For example, simple alkanes, alkenes, and cycloalkanes are readily separated a t temperatures below 150” (Table 111), aromatic compounds a t temperatures between 150-200” (Table IV), compounds of higher molecular weight at temperatures up to 350 “C (Table V). The peaks obtained from compounds as polar as phenols and amines are reasonably symmetrical using volumes ranging from 0.05 to 1.0 p1 (Figure 4). The procedure described in this paper is of interest not only because the packing materials show a high thermal stability ( I ) and may be used to separate complex mixtures of compounds that can otherwise prove intractable on conventional columns (7-9), but also because it is relatively simple to make small, significant and controlled changes in the organic portion of the polysiloxane. For example, from Table 111, it can be readily seen how the introduction of the CC13 group in the polysiloxane makes large changes in the retention times of the alkanes and alkenes studied. Thus, in considering the isomeric pentanes, increased branching lowers the value of the ratio dramatically. A similar trend is noticeable for hexene-1 and 3,3-dimethylbutene-1. On the other hand, there is an opposing trend when considering the number of alkyl substituents adjacent to the carbon double bond in alkenes, and this is illustrated by pentene-1, pentene-2, 2-methylbutene-2, and 2,3-dimethylbutene-2. Again, there are significant changes in the relative retention times for aromatic hydrocarbons on the stationary phases studied (Table IV) and the phases reported in this paper are enabling us to study solute-solvent interactions in closer detail than in many recent studies using existing commercially available liquid phases. This discussion together with results obtained from a wider range of polar and nonpolar polysiloxanes will be given in a subsequent paper.
The Fifth Edition of “Reagent Chemicals” is in press. Meanwhile the Committee on Analytical Reagents is giving advance notification of certain of the requirements for seven reagents included in the Fifth Edition.
Ammonium Molybdate Molybdenum Trioxide Molybdic Acid On pages 65, 378, and 381 in the 4th Edition, raise the limit of the requirement for Arsenate, phosphate, and silicate (as SiOz) from 0.0005% to 0.001%. Add the following separate Phosphate (PO4) requirement: Not more than 5 PPm *
Isopentyl Alcohol On page 302 in the 4th Edition delete the following three requirements: Boiling range, Aldehydes, and Substances darkened by sulfuric acid. Add the four requirements cited below. Assay. Note less than 98.5% Water (HzO). Not more than 0.5%. Acidity (as CH3COOH). Not more than 0.01%. Carbonyl (as HCHO). Not more than 0.1%. Lithium Metaborate (new item) Assay. Not less than 98.0 nor more than 102.0%. Bulk density. Not less than 0.25 g/ml. Insoluble matter. Not more than 0.01%. Loss on fusion a t 950°C. Not more than 2.0%. Aluminium (Al). Not more than 0.001’70. Heavy metals (as Pb). Note more than 0.001%. Iron (Fe). Not more than 0.001’70. Magnesium (Mg). Not more than 5 ppm. Potassium (K). Not more than 0.001~0. Silicon (Si). Not more than 0.004%. Sodium (Na). Not more than 0.001%.
Phenol On page 400 in the 4th Edition, add the following requirement: Freezing point. Not below 405°C (dry basis). Change the requirement for Water (HzO)from “TOpass test (limit about 0.5%)” to “Not more than 0.5%.”
Sodium Carbonate, Anhydrous
ACKNOWLEDGMENT We thank C. B. Thomas for his assistance with the mass spectrometric analyses.
Sodium Carbonate, Alkalimetric Standard
Received for review September 18, 1973. Accepted January 7, 1974. One of us (A.H.A.-T.) is grateful to the Cal-
On pages 519, 523, and 525 in the 4th Edition, lower the requirement for Chlorine (Cl) to 0.001%.
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ANALYTICAL CHEMISTRY, VOL. 46, NO. 8, JULY 1974
Sodium Carbonate, Monohydrate
